Sequence conversion and signal amplifier DNA having poly DNA spacer sequences and detection methods using same

ABSTRACT

Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting said sample, in the presence of a polymerase and an endonuclease, with a sequence conversion oligonucleotide. Also disclosed are methods for detecting a target nucleic acid in a sample in which said sample is contacted, in the presence of a polymerase and an endonuclease, with a sequence conversion oligonucleotide and a signal amplifier oligonucleotide. The disclosure also provides compositions and kits comprising such sequence conversion and signal amplifier oligonucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Application 62/063,666, which was filed on Oct. 14, 2014 andto U.S. Provisional Application 62/098,066, which was filed on Dec. 30,2014. Both U.S. Provisional Applications are incorporated herein byreference in their entireties.

SEQUENCE LISTING

This application contains a Sequence Listing which is incorporated byreference and is submitted with the filing of this application as a textfile titled, “28298US02_SeqList_filed_130 ct2015.txt”. The SequenceListing file was created on Oct. 12, 2015 and is 3,000 bytes in size.

FUNDING

At least a portion of the research disclosed herein was supported by agrant from the Japan Science and Technology Agency (JST), an agency ofthe Government of Japan.

BACKGROUND

The detection of target nucleic acid in test samples is important invarious fields, including medicine and biology. Many compositions, assayplatforms, and procedures are available for the detection of specificnucleic acid molecules. In order for detection to be effective in aclinical environment, these procedures should not only be reproducibleand accurate, but must also be robust and fast.

One common method used for amplification of specific sequences from apopulation of mixed nucleic acid sequences is the polymerase chainreaction (PCR). Since a typical PCR is carried out at three differenttemperatures, the reaction can be associated with challenges such asdifficulty in maintaining accurate temperatures and that the time lossincreases in proportion to the number of amplification cycles. Thedenaturation of a double-stranded template DNA into single strands(while dependent to some extent on the particular sequence) oftenrequires the use of high “melting” temperatures, which limits the classof DNA polymerases that can be used to those that are highlythermostable. Consequently, isothermal amplification platformtechnologies have been developed to detect nucleic acids under reactionconditions that are milder than those used in PCR. Nevertheless, theseisothermal amplification technologies have not addressed the challengesassociated with achieving faster reaction times.

The following disclosure provides alternative methods and compositionsfor detecting a nucleic acid sequence (such as DNA or RNA) underreaction conditions that are less rigorous than those used in PCR. Themethods and compositions maintain sequence selectivity and sensitivitythat allows for the detection of nucleic acid molecules that may be in asample at low concentrations and/or nucleic acid molecules of a shortlength. The methods and compositions also reduce reaction times. Amongother aspects, the disclosure provides novel methods and nucleic acidmolecules that can improve the detection limit of target nucleic acidsin a sample under low temperature, isothermal conditions, and cansimplify or improve sample preparation and automated methods ofdetection, while decreasing reaction times.

SUMMARY OF THE INVENTION

In one aspect, the disclosure relates to a method for detecting a targetnucleic acid in a sample, said method comprising contacting said samplewith: an oligonucleotide (sequence conversion DNA or SC DNA) comprising,in the 5′ to 3′ direction, a signal DNA generation sequence (A), anendonuclease recognition site (B), a poly DNA spacer sequence (PDS), anda sequence (C) complementary to the 3′ end of a target nucleic acid; apolymerase; and an endonuclease for a nicking reaction. In embodimentsof this aspect, the method also comprises determining the presence orabsence of a signal DNA, wherein the presence of the signal DNAindicates the presence of the target nucleic acid in the sample.

In one aspect, the disclosure relates to a method for detecting a targetnucleic acid in a sample, said method comprising contacting said samplewith: a first oligonucleotide (sequence conversion DNA or SC DNA)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(A), an endonuclease recognition site (B), a poly DNA spacer sequence(PDS), and a sequence (C) complementary to the 3′ end of a targetnucleic acid; a second oligonucleotide (signal amplifier DNA or SA DNA)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(D) that is homologous to the signal DNA generation sequence (A) of thefirst oligonucleotide, an endonuclease recognition site (E) (which maybe the same or different from the endonuclease recognition site (B) inthe SC DNA), a poly DNA spacer sequence (PDS) (which may be the same ordifferent from the PDS in the SC DNA), and a sequence (F) that ishomologous to the signal DNA generation sequence (A) of the firstoligonucleotide; a polymerase; and an endonuclease for a nickingreaction. In embodiments of this aspect, the method also comprisesdetermining the presence or absence of a signal DNA, wherein thepresence of the signal DNA indicates the presence of the target nucleicacid in the sample. Also, in some embodiments of this aspect, the signalamplifier DNA (SA DNA) does not have a poly DNA spacer sequence (PDS).

The spacer is a poly “nucleic acid” spacer, and for example can be apoly DNA spacer (PDS), a poly RNA spacer (PRS), or a derivative oranalog thereof (e.g., artificial nucleic acid). In certain embodiments,the poly DNA spacer (PDS) sequence may comprise G, A, T, C, or anycombination thereof. The poly RNA spacer (PRS) sequence may comprise G,A, U, C, or any combination thereof. In addition, the PDS sequence canbe from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 8, from 1 to 6,or from 1 to 5 bases long.

As described in greater detail below, methods disclosed herein areespecially useful for the detection of target RNAs in a sample. Theplacement in a SC DNA of a poly DNA spacer (PDS) sequence between theendonuclease recognition site (B) and the sequence (C) complementary tothe 3′ end of a target greatly enhances the rate at which signal DNA isgenerated. In another aspect, the disclosure relates to the accelerationor enhancement of endonuclease nicking activity downstream from,upstream from, and/or adjacent to RNA-DNA hybrid duplexes.

In certain embodiments the polymerase may have strand displacementactivity. In further embodiments, the polymerase may be 3′ to 5′exonuclease deficient, 5′ to 3′ exonuclease deficient, or both 3′ to 5′exonuclease deficient and 5′ to 3′ exonuclease deficient. In someembodiments the polymerase comprises a DNA polymerase.

In embodiments, the endonuclease may comprise a nicking endonuclease ora restriction endonuclease that can be used in a reaction that nicks anoligonucleotide.

While the method disclosed herein may be performed under typical DNAamplification conditions (e.g., typical temperatures associated withstandard PCR, reactant concentrations, time cycles, etc.), in someembodiments the method may be performed under isothermal conditions orunder substantially constant temperatures. In further embodiments themethod may be performed at temperatures that are lower than temperaturesused in standard PCR methods. As one example, some embodiments of themethod may be performed at a temperature at or below a calculatedoptimal hybridization or annealing temperature, or an experimentallydetermined hybridization or annealing temperature, of the target nucleicacid (T) and the sequence (C) of the SC DNA, or of the signal DNA (S)and the sequence (F) of the SA DNA as described below. In embodiments,the method may be performed at a temperature that is below the meltingtemperature of the target nucleic acid (T) bound to the sequence (C) ofthe SC DNA, or the signal DNA (S) bound to the sequence (F) of the SADNA. In yet other embodiments, the method may be performed attemperatures that allow for polymerase and/or endonuclease activity. Infurther embodiments, the method may be performed at temperatures thatare at or about the optimal reaction temperature for the polymeraseand/or endonuclease present in the reaction mixture for the detection ofa target nucleic acid in a sample.

In another aspect, the disclosure relates to an oligonucleotide, whichmay be referred herein as a “sequence conversion DNA” (or “SC DNA”)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(A), an endonuclease recognition site (B), a poly DNA spacer (PDS)sequence, and a sequence (C) complementary to the 3′ end of a targetnucleic acid.

In another aspect, the disclosure relates to an oligonucleotide, whichmay be referred to herein as a “signal amplifier DNA” (or “SA DNA”)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(D) homologous to a signal DNA generation sequence (A) of a sequenceconversion DNA (SC DNA), an endonuclease recognition site (E), a polyDNA spacer (PDS) sequence, and a sequence (F) which is homologous to asignal DNA generation sequence (A) of a sequence conversion DNA (SCDNA). In yet another aspect, the disclosure relates to signal amplifierDNA comprising, in the 5′ to 3′ direction, a signal DNA generationsequence (D) homologous to a signal DNA generation sequence (A) of asequence conversion DNA (SC DNA), an endonuclease recognition site (E),and a sequence (F) which is homologous to a signal DNA generationsequence (A) of a sequence conversion DNA (SC DNA).

The SC and/or SA DNAs of the present disclosure are generally linear,however these DNAs can also be circular (i.e. mini-circle DNA (mc)).Rolling circle amplification (RCA) can be primed upon binding of the 3′end of a target nucleic acid to a mini-circle SC DNA, or upon binding ofthe 3′ end of a signal DNA to a mini-circle SA DNA. The resulting RCAproduct is a long single-stranded DNA fragment containing thousands ofcopies of the SC DNA or SA DNA.

In one example, the signal DNA generation sequence (A) of a SC DNA canbe complementary to the 5′-end of a target nucleic acid (T). In thisaspect, the target nucleic acid (T) binds to both the signal DNAgeneration sequence (A) and the sequence (C) of the SC DNA. The bindingof target nucleic acid (T) to the SC DNA (in the presence of DNA ligase)results in the formation of a mini-circle SC DNA (mc SC DNA), andsubsequent priming of rolling circle amplification (RCA). The resultingRCA product is a long single-stranded DNA fragment containing thousandsof copies of the SC DNA. In one embodiment the endonuclease recognitionsite of the SC DNA is within the double-stranded stem-loop region of ahairpin structure, and therefore subject to nicking (on the 3′ side ofthe stem-loop) in the presence of a nicking endonuclease. In thepresence of both a nicking endonuclease and polymerase, signal DNA canbe generated directly from the RCA product.

The methods and oligonucleotides of the present disclosure can be usedin combination with other amplification and/or detection schemes. Forexample, any one of the signal DNAs produced in accordance with themethods disclosed herein can serve as a primer in a rolling circleamplification reaction. In one embodiment, the 3′ end of a signal DNAproduced according to methods of the present disclosure can becomplementary to a mini-circle DNA template, and rolling circleamplification can be initiated upon binding of the signal DNA.

The target nucleic acid sequence may be any nucleotide sequence ofinterest and in some embodiments may comprise a sequence that originatesfrom an infectious agent or a micro-RNA. In other embodiments the targetnucleic acid may comprise a sequence from a gene that may be associatedwith a disease or a disorder.

In some embodiments the endonuclease recognition site comprises asequence that is complementary to a sequence that is nicked by anendonuclease. In other embodiments, the sequence that is nicked by theendonuclease is adjacent (downstream or upstream) to the sequence thatis specifically recognized by the endonuclease.

In a further aspect, the disclosure relates to a composition fordetecting a target nucleic acid in a sample, said compositioncomprising: an oligonucleotide (sequence conversion DNA or SC DNA)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(A), an endonuclease recognition site (B), a poly DNA spacer (PDS)sequence, and a sequence (C) complementary to the 3′ end of a targetnucleic acid; a polymerase; and an endonuclease for a nicking reaction.

In a further aspect, the disclosure relates to a composition fordetecting a target nucleic acid in a sample, said compositioncomprising: a first oligonucleotide (sequence conversion DNA or SC DNA)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(A), an endonuclease recognition site (B), a poly DNA spacer (PDS)sequence, and a sequence (C) complementary to the 3′ end of a targetnucleic acid; a second oligonucleotide (signal amplifier DNA or SA DNA)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(D) that is homologous to the signal DNA generation sequence (A) of thefirst oligonucleotide, an endonuclease recognition site (E) (which maybe the same or different from the endonuclease recognition site (B) inthe SA DNA), a poly DNA spacer (PDS) sequence (which may be the same ordifferent from the PDS sequence of the first oligonucleotide), and asequence (F) that is homologous to the signal DNA generation sequence(A) of the first oligonucleotide; a polymerase; and an endonuclease fora nicking reaction. In some embodiments of this aspect, the secondoligonucleotide or signal amplifier DNA (SA DNA) does not have a polyDNA spacer (PDS) sequence.

The compositions can also comprise a polymerase, and/or an endonucleasecapable of nicking at or adjacent to the endonuclease recognition siteof the first and second oligonucleotide when the endonucleaserecognition site is double stranded. Compositions can also include otherreagents such as reaction buffers, deoxyribonucleotides, and reportermolecules such as, for example, fluorophore-modified probe DNAs (e.g.,molecular beacon probes) for the fluorescent detection of newlysynthesized DNA. Reporter molecules generally known in the art can beused, including acridinium conjugated probes (i.e. chemiluminescenttechnology).

In yet another aspect, the disclosure relates to a kit for detecting atarget nucleic acid in a sample, said kit comprising: an oligonucleotide(sequence conversion DNA or SC DNA) comprising, in the 5′ to 3′direction, a signal DNA generation sequence (A), an endonucleaserecognition site (B), a poly DNA spacer (PDS) sequence, and a sequence(C) complementary to the 3′ end of a target nucleic acid; a polymerase;and an endonuclease for a nicking reaction. In some embodiments the kitscan further comprise a polymerase and/or an endonuclease capable ofnicking an endonuclease recognition site or a site adjacent to anendonuclease recognition site. The kits can also include reagents suchas reaction buffers, deoxyribonucleotides, and reporter molecules suchas, for example, fluorophore-modified probe DNAs (e.g., molecular beaconprobes) for the fluorescent detection of newly synthesized DNA such as asignal DNA. The kits can also comprise instructions for use in thepractice of any one of the methods disclosed herein.

In yet another aspect, the disclosure relates to a kit for detecting atarget nucleic acid in a sample, said kit comprising: a firstoligonucleotide (sequence conversion DNA or SC DNA) comprising, in the5′ to 3′ direction, a signal DNA generation sequence (A), anendonuclease recognition site (B), a poly DNA spacer (PDS) sequence, anda sequence (C) complementary to the 3′ end of a target nucleic acid; asecond oligonucleotide (signal amplifier DNA or SA DNA) comprising, inthe 5′ to 3′ direction, a signal DNA generation sequence (D) that ishomologous to the signal DNA generation sequence (A) of the firstoligonucleotide, an endonuclease recognition site (E) (which may be thesame or different from the endonuclease recognition site (B) in the SADNA), a poly DNA spacer (PDS) sequence (which may be the same ordifferent from the PDS sequence of the first oligonucleotide), and asequence (F) that is homologous to the signal DNA generation sequence(A) of the first oligonucleotide. In some embodiments, the secondoligonucleotide or signal amplifier DNA (SA DNA) does not have a polyDNA spacer (PDS) sequence. In some embodiments the kits can furthercomprise a polymerase and/or an endonuclease capable of nicking anendonuclease recognition site or a site adjacent to an endonucleaserecognition site. The kits can also include reagents such as reactionbuffers, deoxyribonucleotides, and reporter molecules such as, forexample, fluorophore-modified probe DNAs (e.g., molecular beacon probes)for the fluorescent detection of newly synthesized DNA such as a signalDNA. The kits can also comprise instructions for use in the practice ofany one of the methods disclosed herein.

The methods, oligonucleotides, compositions, and kits disclosed hereinmay be used in combination with integrated system platforms. Forexample, methods, oligonucleotides, compositions, and kits of thepresent invention may be used in combination Abbott's ARCHITECT system.The methods, oligonucleotides, compositions, and kits disclosed hereinmay be used with sample preparation system platforms such as, forexample, the m2000sp sample preparation system (Abbott Diagnostics,Abbott Park, Ill.). Similarly, the methods, oligonucleotides,compositions, and kits disclosed herein may be used with point-of-caresystem platforms such as, for example, Abbott's i-STAT point-of-caresystem (Abbott Diagnostics, Abbott Park, Ill.). Further, the methods,oligonucleotides, compositions, and kits of the present invention can beused with any number of other devices, assay platforms, andinstrumentation such as, for example, hand held fluorescence detectors,micro-pH meters, microfluidic devices, microarrays, enzymatic detectionsystems, immunochromatographic strips, and lateral flow devices.

The methods, oligonucleotides, compositions, and kits disclosed hereinmay be used in the field of molecular diagnostics, including diagnosisof non-infectious and infectious diseases. For example, methods,oligonucleotides, compositions, and kits of the present invention can beused to detect microRNA, messenger RNA, non-coding RNA and methylatedDNA in human fluid such as blood, urine, saliva, sweat and feces.Similarly, methods, oligonucleotides, compositions, and kits of thepresent invention can be used to detect target nucleic acids originatingfrom infectious diseases such as, for example, HBV, HCV, HIV, HPV,HTLV-I, Parvo virus, Tuberculosis, Syphilis, Malaria and Entamoebahistolytica in human fluid like blood, urine, saliva, sweat and feces.

It is understood that in some aspects of the present disclosure, the SCand SA DNAs disclosed herein may comprise chemically modifiednucleotides. For example, the SC and SA DNAs disclosed herein maycomprise LNA (Locked Nucleic Acid), BNA (Bridged Nucleic Acid), ENA(Ethylene Bridged Nucleic Acid), GNA (Glycol Nucleic Acid), TNA (ThreoseNucleic Acid), PNA (Peptide Nucleic Acid), Morpholino Nucleic Acid,phosphorothioate nucleotides, or combinations and/or derivativesthereof.

Additional aspects, embodiments, and advantages provided by thedisclosure will become apparent in view of the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram schematically illustrating a non-limiting exampleof a Sequence Conversion DNA (SC DNA) for the detection of a targetnucleic acid in a sample. The SC DNA comprises, in the 5′ to 3′direction, a signal generation sequence (A), an endonuclease recognitionsite (B) that can be used in a nicking reaction, a poly DNA spacer (PDS)sequence, and a sequence (C) complementary to the target nucleic acid.

FIG. 1B is a diagram schematically illustrating a non-limiting exampleof a Signal Amplifier DNA (SA DNA) for the detection of a target nucleicacid in a sample. The SA DNA comprises, in the 5′ to 3′ direction, asignal DNA generation sequence (D) homologous to the signal DNAgeneration sequence (A) of a SC DNA; an endonuclease recognition site(E) (which may be the same or different from the endonucleaserecognition site (B) of a SC DNA), a poly DNA spacer (PDS) sequence(that may the same or different from the PDS sequence of a SC DNA), anda sequence (F) that is homologous to the signal DNA generation sequence(A) of a first SC DNA. In some embodiments, the signal amplifier DNA (SADNA) does not have a poly DNA spacer (PDS) sequence.

FIG. 2A is a diagram schematically illustrating the progression of anexemplary reaction of a target (T) nucleic acid with a SequenceConversion (SC) DNA for the detection of a target nucleic acid in asample. Sequences (A)-(C), and the PDS, are as described in FIG. 1A,sequence (T) represents a target sequence, sequence (X) represents thesequence produced when Target (T) bound to sequence (C) is extended bypolymerase, sequence (X′) represents the nicked extension sequence, andsequence (S) represents the signal DNA sequence eventually produced.

FIG. 2B is a diagram schematically illustrating the progression of anexemplary reaction of a signal DNA (S) with a Signal Amplification (SA)DNA for the detection of a target nucleic acid in a sample. Sequences(D)-(F), and the PDS, are as described in FIG. 1B, sequence (S) is theSignal DNA produced from reaction of Target (T) nucleic acid with SC DNAas described in FIG. 2A, sequence (Y) represents the sequence producedwhen Signal DNA (S) bound to sequence (D) is extended by polymerase,sequence (Y′) represents the nicked extension sequence, and sequence (S)represents the signal DNA sequence eventually produced. Because the SAsignal generation sequence (D) is homologous to the SC signal generationsequence (A), the same signal DNA (S) is produced.

FIG. 3 shows the results of the those reactions performed in Example 1,demonstrating that the presence of a PDS sequence positioned between theendonuclease recognition site (B) and the sequence (C) of a SC DNAaccelerates the amplification of signal DNA.

FIG. 4 shows the results of those reactions performed in Example 2,demonstrating that the presence of a PDS sequence positioned between theendonuclease recognition site (B) and the sequence (C) of a SC DNAenhances nicking of endonuclease recognition site (B).

DETAILED DESCRIPTION

In a general sense, the disclosure relates to nucleic acid constructsthat are surprisingly effective in the detection of target nucleic acidsin a test sample. The constructs disclosed herein comprise nucleic acidsequences that allow the production of signal DNAs that are generated inthe presence of a target nucleic acid, with a concomitant increase inthe speed of the reaction. The methods and nucleic acid constructsdisclosed herein provide for selective, sensitive, and fast detection oftarget nucleic acids that may be advantageously performed under lowtemperature and isothermal conditions.

In an aspect, the disclosure relates to an oligonucleotide, which may bereferred to herein as a “signal amplifier DNA” (or “SA DNA”) comprising,in the 5′ to 3′ direction, a first sequence that is complementary to aknown signal DNA sequence, an endonuclease recognition site, a poly DNAspacer (PDS) sequence, and a second sequence that is complementary tothe same known signal DNA sequence as the first sequence. The firstsequence is the signal DNA generation sequence (D) in FIG. 1B, that ishomologous to a known signal DNA generation sequence (A) of a SC DNA.The second sequence is sequence (F) in FIG. 1B, that is homologous tothe same known signal DNA generation sequence (A) of the same SC DNA.

In this aspect, the lengths of the first and second sequences may vary,but typically each of the sequences is about the same length as theother. In embodiments, the length of the sequences may be in a rangefrom about 5 to about 100 nucleotides, but are more typically from about5 to about 30, from about 10 to about 30, or from about 15 to about 30nucleotides in length. The endonuclease recognition site comprises asequence that can be recognized, bound, and nicked by an endonuclease asdescribed herein. Such sequences are generally known in the art. Theendonuclease recognition site can comprise additional nucleotides either5′ or 3′ to the endonuclease binding site (or both 5′ and 3′) but istypically no more than 10 nucleotides in length.

The disclosure provides novel Sequence Conversion (SC) and SignalAmplifier (SA) oligonucleotide constructs, and combinations thereof,that are useful in detecting a target nucleic acid in a sample, with aconcomitant increase in the speed of the reaction. As depicted by theillustrative embodiment of FIG. 1A, a Sequence Conversion DNA (SC DNA)oligonucleotide for the detection of a target nucleic acid in a samplecomprises, in the 5′ to 3′ direction, a signal DNA generation sequence(A), an endonuclease recognition site (B), a poly DNA spacer (PDS)sequence, and a sequence (C) complementary to the 3′ end of a targetnucleic acid.

As depicted by the illustrative embodiment of FIG. 1B, a SignalAmplifier DNA (SA DNA) for the detection of a target nucleic acid in asample comprises, in the 5′ to 3′ direction, a signal DNA generationsequence (D) homologous to the signal DNA generation sequence (A) of aSC DNA; an endonuclease recognition site (E) (which may be the same ordifferent from an endonuclease recognition site (B) of a SC DNA), a polyDNA spacer (PDS) sequence (which may be the same or different from thePDS of a SC DNA), and a sequence (F) comprising a sequence that ishomologous to the signal DNA generation sequence (A) of a SC DNA. Insome embodiments, the signal amplifier DNA (SA DNA) does not have a polyDNA spacer (PDS) sequence.

As illustrated in FIG. 1A, the SC DNAs disclosed herein comprise asignal generation sequence (A). The signal generation sequence (A) inthe SC DNA can comprise any desired nucleic acid sequence and is notlimited by any particular sequence. As discussed in greater detailbelow, the signal generation sequence (A) provides at least a portion ofthe template for a signal DNA (e.g., nucleic acid (S) in FIG. 2), theproduction of which indicates the presence of target nucleic acid. Thesignal generation sequence (A) in the SC DNA is not limited by length.In some embodiments, the signal generation sequence (A) in the SC DNA isfrom about 5 to about 100 nucleic acid bases, and all integers between 5and 100. In embodiments, the signal generation sequence (A) in the SCDNA is from about 5 to about 30 nucleic acid bases, and all integersbetween 5 and 30. In some embodiments, the signal generation sequence(A) in the SC DNA is from about 10 to about 30 nucleic acid bases, andall integers between 10 and 30. In yet further embodiments, the signalgeneration sequence (A) in the SC DNA is from about 15 to about 30nucleic acid bases, and all integers between 15 and 30 (e.g., about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29 or about 30bases).

As illustrated in FIG. 1B, the SA DNAs disclosed herein comprise asignal DNA generation sequence (D) which is homologous to the signal DNAgeneration sequence (A) of a SC DNA, and a sequence (F) which ishomologous to the same signal DNA generation sequence (A) of the same SCDNA. In some embodiments, in order to be homologous to the signal DNAgeneration sequence (A) of a SC DNA, sequences (D) and (F) arecompletely identical to the corresponding signal DNA generation sequence(A). In other embodiments, sequence (F) is identical in sequence to thecorresponding signal DNA generation sequence (A) of a SC DNA, exceptthat it is from about 1 to about 5, or from about 1 to about 4, or fromabout 1 to about 3, or from about 1 to about 2, or 1 base(s) shorter atthe 3′ end. When the signal DNA generation sequence (D) of a SA DNA ishomologous to the signal DNA generation sequence (A) of a SC DNA, itfollows that the same signal DNA (S) is produced and exponentiallyamplified.

The SC and SA DNAs comprise endonuclease recognition sites (B) and (E)respectively, which can be the same or different. In single strandedform (e.g., the structure of FIGS. 1A and 1B) the endonucleaserecognition sites (B) and (E) may comprise a sequence that iscomplementary to a sequence that may be nicked by an endonuclease. Thesequence that is nicked by the endonuclease may be within, downstream,or upstream from the sequence that is recognized by the endonuclease.Suitably, when double stranded, the endonuclease recognition sites (B)and (E) can be recognized by one or more endonucleases present in thereaction, and the endonuclease recognition sites (B) and (E) (or asequence adjacent to the endonuclease recognition sites (B) and (E)) maybe cleaved on only one strand of the double-stranded DNA (i.e., nicked).As described in greater detail below, binding of a target nucleic acidto the complementary sequence (C) of the SC DNA primes replication viaDNA polymerase to create an active, double-stranded form of theendonuclease recognition site (B) that can now serve as a recognitionsite for an endonuclease (FIG. 2A). Endonuclease nicking at the newlycreated double-stranded endonuclease site (B), or at a site adjacent tonewly created double-stranded endonuclease site (B), then primesreplication via DNA polymerase and generates signal DNA (S) (see, e.g.,FIG. 2A). As illustrated in FIG. 2A, the endonuclease recognition site(B) is oriented such that the newly replicated strand is nicked, not theSC DNA. That is, when the newly replicated strand is generated theorientation of the endonuclease recognition site in (B) directsendonuclease activity (cleavage) of the newly replicated strand. Assuch, the endonuclease recognition site comprises a sequence that iscomplementary to a sequence that is nicked by an endonuclease, allowingthe SC oligonucleotide to remain intact throughout the reaction (i.e.,the SC DNA is not nicked or cleaved).

The SC and SA DNAs can also comprise a poly DNA spacer (PDS) sequence.In the SC DNA, the PDS sequence is positioned between the endonucleaserecognition site (B) and the sequence (C) complementary to the 3′ end ofthe target nucleic acid. In the SA DNA, the PDS sequence is positionedbetween the endonuclease recognition site in (E) and the sequence (F)substantially homologous to the signal generation sequence (A) of a SCDNA. In some embodiments, the signal amplifier DNA (SA DNA) does nothave a poly DNA spacer (PDS) sequence. The PDS sequence of the SC and SADNAs can be the same or different. The PDS sequence can be comprised ofany one or more of the natural bases G, A, T or C, and can be from 3 to20, from 3 to 18, from 3 to 16, from 3 to 14, from 3 to 14, from 3 to12, from 3 to 10, from 3 to 8, from 3 to 6, or from 3 to 5 bases long.As illustrated below, the presence of a PDS sequence in one or more ofthe SC or SA DNAs disclosed herein can accelerate reactions performedaccording to the methods disclosed herein by as much as 5 to 60, 5 to50, 5 to 40, 5 to 30, 5 to 20, or 5 to 10 minutes.

As described in greater detail below, binding of signal DNA (S),generated from the signal generation sequence (A) of a SC DNA, to thesequence (F) of a SA DNA primes replication via DNA polymerase to createan active, double-stranded form of the endonuclease recognition site (E)of the SA DNA that can serve as a recognition site for an endonuclease(FIG. 2B). Endonuclease nicking at the newly created double-strandedendonuclease site (E) of the SA DNA, or at a site adjacent to newlycreated double-stranded endonuclease site (E), then primes replicationvia DNA polymerase and generates signal DNA (S) that is the same assignal DNA (S) generated from the SC DNA (FIG. 2B). As illustrated inFIG. 2B, the endonuclease recognition site (E) is oriented such that thenewly replicated strand is nicked, not the SA DNA. That is, when thenewly replicated strand is generated the orientation of the endonucleaserecognition site in E directs endonuclease activity (cleavage) of thenewly replicated strand. As such, the endonuclease recognition sitecomprises a sequence that is complementary to a sequence that is nickedby an endonuclease, allowing the SA oligonucleotide to remain intactthroughout the reaction (i.e., the SA DNA is not nicked or cleaved).

The sequence (C) of the SC DNA that is complementary to the target DNAis not limited by length, and can be from about 5 to about 100 nucleicacid bases, and all integers between 5 and 100. In some embodiments, thesequence (C) of the SC DNA is from about 5 to about 30 nucleic acidbases, and all integers between 5 and 30. In some embodiments, thesequence (C) in the SC DNA is from about 10 to about 30 nucleic acidbases, and all integers between 10 and 30. In further embodiments, thesequence (C) of the SC DNA is from about 15 to about 30 nucleic acidbases, and all integers between 15 and 30.

Complementary sequences are capable of forming hydrogen bondinginteractions to form a double stranded nucleic acid structure (e.g.,nucleic acid base pairs). For example, a sequence that is complementaryto a first sequence includes a sequence which is capable of formingWatson-Crick base-pairs with the first sequence. As used herein, theterm “complementary” does not require that a sequence is complementaryover the full-length of its complementary strand, and encompasses asequence that is complementary to a portion of another sequence. Thus,in some embodiments, a complementary sequence encompasses sequences thatare complementary over the entire length of the sequence or over aportion thereof (e.g., greater than about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% ofthe length of the sequence). For example, two sequences can becomplementary to each other over a length ranging from about 2 to about100 consecutive (contiguous) nucleotides, or any integer between 2 and100. In some embodiments, two sequences can be complementary to eachother over a length ranging from about 15 to about 30 consecutive(contiguous) nucleotides, or any integer between 15 and 30. As usedherein, complementary sequences can encompass sequences that have somesequence mismatches. For example, complementary sequences can includesequences that are complementary to at least about 70% to 100%,preferably greater than above 95% of the length of the sequence. Despitesome amount of mismatches, complementary sequences generally have theability to selectively hybridize to one another under appropriateconditions such as, for example, stringent and highly stringentconditions such as those described herein or generally known by those ofordinary skill in the art.

The SC and SA DNAs may be synthesized by known methods. For example, theSC and SA DNAs can be synthesized using a phosphoramidite method, aphosphotriester method, an H-phosphonate method, or a thiophosphonatemethod. In some embodiments, the SC and/or SA DNAs can be purified, forexample using ion exchange HPLC.

The SC and SA DNAs may comprise chemical modifications such as aregenerally known in the art. In some embodiments, for example, the SC andSA DNAs can comprise chemically modified nucleotides (e.g., 2′-0 methylderivative, phosphorothioates, etc.), 3′ end modifications, 5′ endmodifications, or any combinations thereof. In some embodiments, the 3′end of the SC and SA DNAs may be modified such that an extensionreaction does not occur from the 3′ end of the SC or SA DNA (e.g., uponbinding of a target sequence, or another non-target sequence, that mightserve as a primer for polymerase extension). As illustrated in FIG. 2A,it is the 3′ end of the target nucleic acid (T), not the SC DNA, whichinitiates DNA replication. Any replication initiated from the 3′ end ofthe SC or SA DNAs may lead to detection errors (e.g., false positives).Further, non-specific extension reactions from an unmodified 3′ end ofthe SC DNA arising from events such as, for example, binding between theSC DNA and a non-target sequence, binding between the SC DNA and atarget sequence at an incorrect position, binding between SC and SADNAs, or non-templated de novo or ab initio DNA synthesis may also leadto detection errors. Accordingly, in embodiments, the SC and SA DNAscomprise a 3′ end modification that can reduce or eliminate theoccurrence of any non-desired extension reactions, such as thosediscussed above. Non-limiting examples of 3′-end modifications includeTAMRA, DABCYL, and FAM. Other non-limiting examples of modificationsinclude, for example, biotinylation, fluorochromation, phosphorylation,thiolation, amination, inverted nucleotides, or abasic groups.

In another aspect, the present invention encompasses methods fordetecting a target nucleic acid (T) in a sample. The methods generallycomprise contacting said sample with: a first oligonucleotide (orsequence conversion DNA or SC DNA) comprising, in the 5′ to 3′direction, a signal DNA generation sequence (A), an endonucleaserecognition site (B), a poly DNA spacer (PDS) sequence, and a sequence(C) which is complementary to the 3′ end of said target nucleic acid(T); a second oligonucleotide (or signal amplifier DNA or SA DNA)comprising, in the 5′ to 3′ direction, a signal DNA generation sequence(D) homologous to the signal DNA generation sequence (A) of the firstoligonucleotide, an endonuclease recognition site (E) (which is the sameas the endonuclease recognition site (B) of the first oligonucleotide),a poly DNA spacer (PDS) sequence (which is the same or different fromthe PDS of the first oligonucleotide), and a sequence (F) which ishomologous to the signal DNA generation sequence (A) of the firstoligonucleotide; a polymerase; and an endonuclease for a nickingreaction. In embodiments of this aspect, the method also comprisesdetermining the presence or absence of a signal DNA, wherein thepresence of the signal DNA indicates the presence of the target nucleicacid in the sample. In some embodiments, the signal amplifier DNA (SADNA) does not have a poly DNA spacer (PDS) sequence.

The method comprises contacting a sample with an endonuclease. Theendonuclease may be a nicking endonuclease or a restriction endonucleasethat is capable of or that can be used in nicking the sequencecomplementary to the endonuclease recognition site (B) within the SCDNA, or the sequence complementary to the endonuclease recognition site(E) within the SA DNA. In some embodiments, the endonuclease comprises anicking endonuclease or a restriction endonuclease that can catalyze orcan be used to catalyze a double-stranded DNA nicking reaction. Inembodiments providing a nicking endonuclease, the phosphodiester linkageof one strand of a double-strand DNA may be cleaved to generate aphosphate group on the 5′ side of the cleavage site and a hydroxyl groupon the 3′ side. Non-limiting examples of nicking endonucleases includeNb.BbvCI, Nt.AlwI, Nt.BbvCI, Nb.BsrDI, Nb.Btsl, Nt.BspQI, Nt.BstNBI,Nb.BsmI, Nt.C viPII, and Nt.BsmAI.

In some embodiments, the endonuclease may be a restriction endonuclease.In these embodiments the restriction endonuclease recognition site maybe modified so that the restriction endonuclease cleaves thephophodiester bond on only one strand of a double stranded DNA, andgenerates a nick in the double strand. Methods or strategies may be usedto modify the activity of the restriction endonuclease such as, forexample, including a chemical modification in at least one strand of adouble-stranded nucleic acid that is not cleaved by the restrictionenzyme. One non-limiting example of such a modification includesreplacing the oxygen atom of phosphodiester linkage of one strand with asulfur atom.

In embodiments providing a restriction endonuclease, the phosphodiesterlinkage of one strand of a double-strand DNA may be cleaved to generatea phosphate group on the 5′ side of the cleavage site and a hydroxylgroup on the 3′ side. Non-limiting examples of restriction endonucleasesinclude Hinc II, Hind II, Ava I, Fnu4HI, Tth111I and NciI.

The method comprises contacting a sample with a polymerase. In someembodiments, the polymerase may be a DNA polymerase having stranddisplacement activity. In some embodiments, the polymerase may be apolymerase that lacks 5′-3′ exonuclease activity, lacks 3′-5′exonuclease activity, or lacks both 5′-3′ and 3′-5′ exonucleaseactivity. The polymerase may be eukaryotic, prokaryotic, or viral inorigin, and can also be genetically modified. In some embodiments, thepolymerase is selected from among those that function at lowertemperatures, including ambient (e.g., room) temperatures. Non-limitingexamples of DNA polymerases include Klenow fragments, DNA polymerase Iderived from E. coli, 5′ to 3′ exonuclease-deficient Bst DNA polymerasesderived from Bacillus stearothermophilus, and 5′ to 3′exonuclease-deficient Bca DNA polymerases derived from Bacilluscaldotenax.

One non-limiting embodiment of the methods disclosed herein isillustrated in FIGS. 2A and 2B. Briefly, as illustrated in FIG. 2A, asample is contacted with SC DNA in the presence of a DNA polymerase andan endonuclease capable of nicking the double-stranded form (i.e.,complementary sequence) of the endonuclease recognition site (B), or asite adjacent to the double-stranded form of the endonucleaserecognition site (B). If a target nucleic acid (T) is present in thesample, the 3′ end sequence of the target nucleic acid (T) hybridizes tothe sequence (C) of the SC DNA which is complementary to the target andprimes or initiates replication (by the DNA polymerase present in thereaction mixture) thereby generating double stranded extension sequence(X) that includes the double stranded endonuclease recognition site (B).Recognition of the newly-generated double stranded endonucleaserecognition site (B) (by the endonuclease present in the reactionmixture), and subsequent nicking of the newly-generated strand (by theendonuclease present in the reaction mixture), generates oligonucleotidesignal sequence (S) and extension sequence (X′). Because the 3′-OH ofsequence (X′) at the nick serves as an initiation site for subsequentrounds of strand displacement replication, oligonucleotide (S) isdisplaced from the SC DNA by DNA polymerase which continues to replicateand amplify signal DNA (S) in the reaction mixture.

As further illustrated in FIG. 2B, the signal resulting from theproduction of signal DNA (S) can be further amplified by the presence ofa signal amplifier DNA (or SA DNA). Briefly, signal DNA (S) present in areaction hybridizes to the sequence (F) of the SA DNA which primes orinitiates replication (by the DNA polymerase present in the reactionmixture) thereby generating double stranded extension sequence (Y) thatincludes the double stranded endonuclease recognition site (E).Recognition of the newly-generated double stranded endonucleaserecognition site (E) (by endonuclease present in the reaction mixture),and subsequent nicking of the newly-generated strand (by endonucleasepresent in the reaction mixture), generates oligonucleotide signalsequence (S) and extension sequence (Y′). Because the 3′-OH of sequence(Y′) at the nick serves as an initiation site for subsequent rounds ofstrand displacement replication, oligonucleotide (S) is displaced fromthe SA DNA by DNA polymerase which continues to replicate and amplifysignal DNA (S) in the reaction mixture.

Methods according to the present invention are useful for the detectionof target nucleic acids in a sample. In particular, methods of thepresent invention are useful for the detection of target RNA in asample. As exemplified below, the presence in a SC DNA of a poly DNAspacer (PDS) sequence between the endonuclease recognition site (B) andthe sequence (C) complementary to the 3′ end of a target nucleic acidenhances the production of signal DNA (S). As also exemplified below,the presence of the PDS enhances the rate at which endonuclease nicksthe endonuclease recognition site (B).

Methods according to the invention may be performed under isothermal orsubstantially constant temperature conditions. In embodiments thatrelate to performing the method under a substantially constanttemperature, some fluctuation in temperature is permitted. For example,in some embodiments a substantially constant temperature may fluctuatewithin a desired or identified target temperature range (e.g., about+/−2° C. or about +/−5° C.). In embodiments, a substantially constanttemperature may include temperatures that do not include thermalcycling. In some embodiments, methods can be performed at isothermal orsubstantially constant temperatures such as, for example, (1)temperatures at or below about the calculated/predicted orexperimentally determined optimal hybridization or annealing temperatureof the target nucleic acid (T) to sequence (C) of the SC DNA; (2)temperatures at or below the melting temperature of the target nucleicacid (T) bound to SC DNA (typically, hybridization or annealingtemperatures are slightly below the melting temperature); (3)temperatures at or below the melting temperature of the signal DNA (S)bound to SA DNA; or (4) temperatures at or about thecalculated/predicted or experimentally determined optimal reactiontemperature for the polymerase and/or endonuclease present in thereaction mixture.

The methods may comprise reaction temperatures that range from about 20°C. to about 70° C., including lower temperatures falling within therange of about 20° C. to about 42° C. In some embodiments, the reactiontemperature range is from 35° C. to 40° C. (e.g., 35° C., 36° C., 37°C., 38° C., 39° C., or 40° C.). In other embodiments, the reactiontemperature is below 65° C., including lower temperatures below about55° C., about 50° C., about 45° C., about 40° C., or about 30° C. Instill other embodiments, reaction temperatures may be about 20° C.,about 21° C., about 22° C., about 23° C., about 24° C., about 25° C.,about 26° C., about 27° C., about 28° C., about 29° C., about 30° C.,about 31° C., about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., about 40° C.,about 41° C., about 42° C., about 43° C., about 44° C., about 45° C.,about 46° C., about 47° C., about 48° C., about 49° C., about 50° C.,about 51° C., about 52° C., about 53° C., about 54° C., about 55° C.,about 56° C., about 57° C., about 58° C., about 59° C., about 60° C.,about 61° C., about 62° C., about 63° C., about 64° C., about 65° C.,about 66° C., about 67° C., about 68° C., about 69° C., or about 70° C.

The methods may be performed for a time that is adequate to allow foramplification of a detectable amount of signal sequence in the presenceof a target nucleic acid. In some embodiments, the reaction time mayrange from about 5 minutes to 16 hours, or from about 3 minutes to 16hours. In still other embodiments, the reaction time may range fromabout 5 to 120 minutes, or from about 15 to 60 minutes.

Throughout the specification, oligonucleotide (S) is also referred to asa signal DNA (S). Because signal DNA is generated only in the presenceof the target nucleic acid (T), methods according to the presentinvention detect the presence or absence of a target nucleic acid (T) ina sample by detecting the presence or absence of signal DNA. The signalDNA (S) is not limited by sequence, and can be any sequence that isamenable to detection. The signal DNA is also not limited by length.Preferably, the signal DNA can be from about 5 to about 100 bases, andany integer between 5 and 100. In some embodiments, the signal DNA canbe from about 5 to about 30 nucleic acid bases, and all integers between5 and 30. In some embodiments, the signal DNA can be from about 10 toabout 30 bases in length and all integers between 10 and 30. In yetfurther embodiments, the signal DNA can be from about 15 to about 30bases in length and all integers between 15 and 30.

Methods according to the disclosure may be performed under bufferconditions that comprise a pH range from about 4 to about 10, or fromabout 7 to about 9. The buffer may comprise a salt concentration fromabout 10 mM to about 500 mM, or from about 50 mM to 150 mM. In someembodiments the method may be performed using an amount of SC and/or SADNAs that allows for amplification of a detectable amount of signalsequence in the presence of a target nucleic acid. In some embodiments,the SC and/or SA DNA concentration may range from about 100 μM to about100 μM, from about 1 nM to about 1 μM, from about 5 nM to about 50 nM,or from about 5 nM to about 25 nM.

The presence of signal DNA (S) can be detected by any method known inthe art. For example, gel electrophoresis and staining with ethidiumbromide can be used. Also, the presence of signal DNA can be detectedusing fluorescence polarization, immunoassay, fluorescence resonanceenergy transfer, enzyme labeling (such as peroxidase or alkalinephosphatase), fluorescent labeling (such as fluorescein or rhodamine),chemiluminescence, bioluminescence, surface plasmon resonance (SPR), ora fluorophore-modified probe DNA (e.g., TaqMan probe). The amplificationproduct can also be detected by using a labeled nucleotide labeled witha biotin, for example. In such a case, the biotin in the amplificationproduct can be detected using fluorescence-labeled avidin orenzyme-labeled avidin, for example. The amplification product can alsobe detected with electrodes by using redox intercalator known to thoseskilled in the art. The amplification product can also be detected usingsurface plasmon resonance (SPR), a Quarts Crystal Microbalance (QCM), orelectrochemical methods (including those methods employing nanoporesensors).

The methods according to the present invention detect the presence orabsence of a target nucleic acid (T) in a sample. The methods accordingto the present invention can also be used to quantitatively measure theconcentration of a target nucleic acid in a test sample. For example,methods according to the present disclosure can be performed in thepresence of a range of different known concentrations of the targetnucleic acid, and calibration curves can then be prepared and used asgenerally practiced in the art. The target nucleic acid ((T) in FIG. 2)can comprise any nucleic acid sequence and can include DNA, RNA,chemically modified nucleic acids, non-natural nucleic acids, nucleicacid analogs, or any hybrid or combination thereof. Accordingly, in someembodiments, DNA may include cDNA, genomic DNA, and synthetic DNA, andRNA may include total RNA, mRNA, rRNA, siRNA, hnRNA, piRNA, aRNA, miRNA,and synthetic RNA. While some embodiments relate to particular targetnucleic acid sequences, any nucleic acid sequence, including auxiliarynucleic acid sequence, can be a target nucleic acid sequence to bedetected. The disclosure provides for the detection of a target nucleicacid with selectivity and sensitivity even when the nucleic acid is ashort-chain nucleic acid. Accordingly, the degree of complementaritybetween sequences (C) of the SC DNA and target nucleic acid (T) allowsfor specific hybridization between the sequences (e.g., the number ofcomplementary nucleotides in sequence (C) of the sequence conversion DNAand target nucleic acid (T) sequences avoids non-specific hybridizationunder a given set of reaction conditions).

In embodiments, the target nucleic acid sequence can be from, or derivedfrom any number of sources including, for example, genomic DNA,expressed mRNA, nucleic acid sequences from pathogens (microbes,viruses), or therapeutic nucleic acids. Accordingly, the SC and SA DNAsand the methods disclosed herein may be used for the diagnosis andprognosis of diseases (e.g., arising from genetic and infectioussources), identification of contaminants (e.g., food-borne illnesses,equipment contamination), personalized medicine (e.g., monitoring and/orprognosis of a therapy), and the like. For example, molecular diagnostictesting can be performed with respect to the following infectiousdiseases: Hepatitis B Virus (HBV); hepatitis C (HCV); HCV (genotypes1-6); Human Immunodeficiency Virus type 1 (HIV-1); Chlamydiatrachomatis; Neisseria gonorrhoeae; influenza A; influenza B;Respiratory Syncytial Virus (RSV); and Parvo virus.

In some embodiments, the target nucleic acid can comprise micro-RNAs(miRNA). Micro-RNAs include small non-coding RNA molecules of about 22nucleotides. Micro-RNAs are known to function in transcription andpost-transcriptional regulation of gene expression. It is known thatmicro-RNAs function by base pairing with complementary regions ofmessenger RNA (mRNA), resulting in gene silencing via translationalrepression or target degradation.

Any type of sample that may comprise a target nucleic acid may be usedin the methods disclosed herein. As such, the sample containing orsuspected of containing a target nucleic acid is not specificallylimited, and includes, for example, biological samples derived fromliving subjects, such as whole blood, serum, buffy coat, urine, feces,cerebrospinal fluid, seminal fluid, saliva, tissue (such as canceroustissue or lymph nodes), cell cultures (such as mammalian cell culturesor bacterial cultures); samples containing nucleic acids, such asviroids, viruses, bacteria, fungi, yeast, plants, and animals; samples(such as food and biological preparations) that may contain or beinfected with microorganisms such as viruses or bacteria; and samplesthat may contain biological substances, such as soil, industrial processand manufacturing equipment, and wastewater; and samples derived fromvarious water sources (e.g., drinking water). Furthermore, a sample maybe processed by any known method to prepare a nucleic acid-containingcomposition used in the methods disclosed herein. Examples of suchpreparations can include cell breakage (e.g., cell lysates andextracts), sample fractionation, nucleic acids in the samples, andspecific nucleic acid molecular groups such as mRNA-enriched samples.The sample used in the method for detecting a target nucleic acid of thepresent invention is not limited to those derived from biological andnatural products as mentioned above and may be a sample containing asynthetic oligonucleotide.

Methods according to the present invention can be performed incombination with the Abbott m2000sp sample preparation system. Them2000sp uses magnetic particle technology to capture nucleic acids andwashes the particles to remove unbound sample components. The boundnucleic acids are eluted and transferred to a 96 deep-well plate. TheAbbott m2000sp can also combine with the washed nucleic acidstransferred to the 96 deep-well plate any reagents required to performthe methods according to the present technology. For example, SC and SADNAs, polymerases, endonucleases, molecular beacons, and any otherreagent (e.g., dNTPs) can be added as required, or desired.

Methods according to the present invention can also be interfaced withpoint-of-care platforms. For example, the incorporation of adeoxyribonucleotide triphosphate (dNTP) into a growing DNA strandinvolves the formation of a covalent bond and the release ofpyrophosphate and a positively charged hydrogen ion affecting the pH ofa reaction. As such, the synthesis of signal DNA according to methods ofthe present invention can be detected by tracking changes in pH using,for example, point-of-care micro-pH meters. For example, Abbott's i-STATpoint-of-care system can be supplied with single-use disposablecartridges containing micro fabricated sensors, calibration solutions,fluidic systems, and waste chambers for analysis of pH.

The methods disclosed herein can comprise additional reagents. Somenon-limiting examples of other reagents that can be used in the nucleicacid amplification reaction include metallic salts such as sodiumchloride, magnesium chloride, magnesium acetate, and magnesium sulfate;substrates such as dNTP mix; and buffer solutions such as Tris-HClbuffer, tricine buffer, sodium phosphate buffer, and potassium phosphatebuffer. Likewise, detergents, oxidants and reducing agents can also beused in the practice of the methods disclosed herein. Furthermore,agents such as dimethyl sulfoxide and betaine (N, N,N-trimethylglycine); acidic substances described in InternationalPublication No. WO 99/54455; and cationic complexes can be used.

The methods and nucleic acid structures provided herein may be used incombination with other methods to provide for the exponentialamplification of a signal DNA in the presence of a target nucleic acid.For example, the methods and compositions according to the presentdisclosure may be used in combination with covered sequence conversionDNAs, as described in U.S. Provisional Application 61/927,710, entitled“Covered Sequence Conversion DNA and Detection Methods” which isincorporated herein by reference.

The term “about” generally refers to a range of numbers that one ofskill in the art would consider equivalent to the recited value (i.e.,having the same function or result). The term “about”, as used herein,is intended to refer to ranges of approximately 10-20% greater than orless than the referenced value. In certain circumstances, one of skillin the art will recognize that, due to the nature of the referencedvalue, the term “about” can mean more or less than a 10-20% deviationfrom that value.

The Examples that follow are intended to be illustrative of the aspectsand embodiments described above. Neither the above disclosure nor theExamples below should be viewed as limiting to the scope of the appendedclaims. One of skill in the art will appreciate that the disclosure isnot limited by the particular terminology which is used to describe andillustrate the various aspects of the disclosure.

Example 1

The following example demonstrates that the presence of a PDS sequencepositioned between the endonuclease recognition site (B) and thesequence (C) of a SC DNA accelerates the amplification of signal DNA.Reactions were performed using SC DNA 1:5′-AGCCCTGTACAATGCCCTCAGCCTGTTCCTGCTGAACTGAGCCA-TAMRA-3′ (SEQ ID NO.:1),and a number of other SC DNAs having different PDS sequences positionedbetween the endonuclease recognition site (B) and the sequence (C) ofthe SC DNA.

These SC DNAs include:

SC DNA SEQ ID NO. SEQUENCE (PDS sequence bolded) 1 15′-AGCCCTGTACAATGCCCTCAGCCTGTT CCTGCTGAACTGAGCCA--TAMRA-3′ T3 25′-AGCCCTGTACAATGCCCTCAGCTTTCT GTTCCTGCTGAACTGAGCCA--TAMRA-3′ T4 35′-AG CCCTGTACAATGCCCTCAGCTTTTC TGTTCCTGCTGAACTGAGCCA--TAMRA-3′ T5 45′-AG CCCTGTACAATGCCCTCAGCTTTTT CTGTTCCTGCTGAACTGAGCCA--TAMRA-3′

The reactions were performed at 37° C. in a 25 μL reaction volumecontaining New England Biolabs (NEB) Buffer 2 having a finalconcentration of 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, pH7.9. The nicking endonuclease used in the reaction was Nb.BbvCI, whichwas present at a concentration of 0.1 units/μL. The polymerase used inthe reaction was Bst DNA Polymerase Large Fragment, which was present ata concentration of 0.08 units/μL. The dNTPs were present at a finalconcentration 200 μM each.

SC DNAs were present in the reaction at a final concentration of 200 nM.The target nucleic acid was human micro RNA 24-3p (SEQ ID NO.:5), andwas present at a concentration of either 1 nM or 100 μM. A MolecularBeacon probe present at a final concentration of 100 nM was used todetect the generation of signal DNA. Fluorescent measurements wereperformed using a Bio-Rad real-time PCR system CFX96, and the resultsare shown in FIG. 3. As illustrated, the presence of a PDS sequencepositioned between the endonuclease recognition site (B) and thesequence (C) of the SC DNA accelerates the amplification of signal DNA.

Example 2

The following example demonstrates that the presence of a PDS sequencepositioned within a SC DNA between the endonuclease recognition site (B)and the sequence (C) complementary to a target nucleic acid acceleratesendonuclease nicking of the endonuclease recognition site (B).

Reactions were performed using the following SC DNA:5′-AGCCCTGTACAATGCCCTCAGCCTGTTCCTGCTGAACTGAGCCA-idT-idT′ (SEQ ID NO.:6).

A number of different target nucleic acids were used, including thefollowing:

Nucleic Acid SEQ ID NO. Sequence DNA #18 7 5′ TGGCTCAGTTCAGCAGGAACAGRNA #6 5 5′ UGGCUCAGUUCAGCAGGAACAG RNA #7 8 5′ UGGCUCAGUUCAGCAGGAARNA #8 9 5′ UGGCUCAGUUCAGCAG

A first polymerization reaction was performed at 37° C. in a 50 μLreaction volume containing New England Biolabs (NEB) Buffer 2 having afinal concentration of 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mMDTT, pH 7.9. The polymerase used in the reaction was Bst DNA PolymeraseLarge Fragment, which was present at a concentration of 0.08 units/μL.The dNTPs were present at a final concentration 200 μM each. SequenceConversion DNA was present at 200 nM. Target nucleic acid (DNA #18, RNA#6, 7, or 8) was present at 200 nM. The polymerization reactions wereincubated at 37° C. for 10 minutes, followed by incubation at 80° C. for20 minutes, after which reactions were moved to 4° C.

12.5 μL of the above polymerization reaction was then transferred to anendonuclease reaction. The endonuclease reaction was performed at 37° C.in a 25 μL reaction volume containing New England Biolabs (NEB) Buffer 2having a final concentration of 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2,1 mM DTT, pH 7.9. The nicking endonuclease used in the reaction wasNb.BbvCI, which was present at a concentration of 0.1 units/μL. Thereactions were incubated for 0 and 5 minutes at 37° C. The reactionswere stopped with the addition of 5 μL of 0.5 M EDTA, followed byincubation at 95° C. A Molecular Beacon probe was used to detect thegeneration of signal DNA, and reaction products were visualized on a 12%TBE-PAGE. (See FIG. 4 (arrow indicating location of small fragmentgenerated from Nb.BbvCI endonuclease nicking).) As shown, when RNA #6was used, no short fragment was generated, demonstrating that thepresence of a PDS sequence in a SC DNA between the endonucleaserecognition site (B) and the sequence (C) complementary to a targetnucleic acid accelerates endonuclease nicking of the endonucleaserecognition site (B).

While the application has been described with reference to certainaspects and embodiments, it will be understood by those skilled in theart that changes may be made to the disclosure provided herein, andequivalents may be substituted without departing from the scope of thedisclosure. Accordingly, the application should not be limited to theparticular aspects and embodiments disclosed, but should be understoodand appreciated to include all aspect and embodiments falling within thescope of the appended claims.

The invention claimed is:
 1. A method for detecting a target nucleicacid in a sample, said method comprising contacting said sample with: afirst oligonucleotide comprising, in the 5′ to 3′ direction, a signalDNA generation sequence, an endonuclease recognition site, a poly DNAspacer sequence, and a sequence complementary to the 3′ end of saidtarget nucleic acid; a second oligonucleotide comprising, in the 5′ to3′ direction, a signal DNA generation sequence homologous to the signalDNA generation sequence of the first oligonucleotide, an endonucleaserecognition site, a poly DNA spacer sequence, and a sequence that ishomologous to the signal DNA generation sequence of the firstoligonucleotide; wherein the poly DNA spacer sequence in the firstoligonucleotide is not part of the endonuclease recognition site or thesequence complementary to the 3′ end of said target nucleic acid, andthe poly DNA spacer sequence in the second oligonucleotide is not partof the endonuclease recognition site or the sequence that is homologousto the signal DNA generation sequence of the first oligonucleotide; andwherein the poly DNA spacer sequence in each of the first and secondoligonucleotide is 3 or more nucleotide bases; a polymerase; and anendonuclease for a nicking reaction; to form a reaction mixture;maintaining the reaction mixture under conditions that produce signalDNA; and detecting the signal DNA, wherein the presence of the signalDNA detects the target nucleic acid.
 2. The method of claim 1, whereinsaid first or second oligonucleotide comprises a poly DNA spacersequence comprising from 3 to 20 bases.
 3. The method of claim 1,wherein said method is performed at a substantially constanttemperature.
 4. The method of claim 1, wherein said method is performedat a temperature of from about 20° C. to about 42° C.
 5. The method ofclaim 1, wherein said polymerase has strand displacement activity, is 3′to 5′ exonuclease deficient, 5′ to 3′ exonuclease deficient, or acombination thereof.
 6. The method of claim 1 wherein said target is amicro-RNA or from an infectious agent.
 7. A composition for detecting atarget nucleic acid in a sample, said composition comprising: a firstchemically modified oligonucleotide comprising, in the 5′ to 3′direction, a signal DNA generation sequence, an endonuclease recognitionsite, a poly DNA spacer sequence, and a sequence complementary to the 3′end of said target nucleic acid; a second chemically modifiedoligonucleotide comprising, in the 5′ to 3′ direction, a signal DNAgeneration sequence homologous to the signal DNA generation sequence ofthe first chemically modified oligonucleotide, an endonucleaserecognition site, a poly DNA spacer sequence, and a sequence that ishomologous to the signal DNA generation sequence of the first chemicallymodified oligonucleotide; wherein the poly DNA spacer sequence in thefirst chemically modified oligonucleotide is not part of theendonuclease recognition site or the sequence complementary to the 3′end of said target nucleic acid, and the poly DNA spacer sequence in thesecond chemically modified oligonucleotide is not part of theendonuclease recognition site or the sequence that is homologous to thesignal DNA generation sequence of the first chemically modifiedoligonucleotide; wherein the poly DNA spacer sequence in each of thefirst and second chemically modified oligonucleotide is 3 or morenucleotide bases; and wherein each of the first and the secondchemically modified oligonucleotide comprise a 3′-terminal chemicalmodification.
 8. The composition of claim 7, wherein said first orsecond oligonucleotide comprises a poly DNA spacer sequence comprisingfrom 3 to 20 bases.
 9. The composition of claim 7, further comprising apolymerase and an endonuclease for a nicking reaction.
 10. Thecomposition of claim 9 wherein said polymerase has strand displacementactivity.
 11. The composition of claim 7 wherein said target nucleicacid is a micro-RNA or from an infectious agent.
 12. A kit for detectinga target nucleic acid in a sample, said kit comprising: a firstchemically modified oligonucleotide comprising, in the 5′ to 3′direction, a signal DNA generation sequence, an endonuclease recognitionsite, a poly DNA spacer sequence, and a sequence complementary to the 3′end of said target nucleic acid; and a second chemically modifiedoligonucleotide comprising, in the 5′ to 3′ direction, a signal DNAgeneration sequence homologous to the signal DNA generation sequence ofthe first chemically modified oligonucleotide, an endonucleaserecognition site, a poly DNA spacer, and a sequence that is homologousto the signal DNA generation sequence of the first chemically modifiedoligonucleotide; wherein the poly DNA spacer sequence in the firstchemically modified oligonucleotide is not part of the endonucleaserecognition site or the sequence complementary to the 3′ end of saidtarget nucleic acid, and the poly DNA spacer sequence in the secondchemically modified oligonucleotide is not part of the endonucleaserecognition site or the sequence that is homologous to the signal DNAgeneration sequence of the first chemically modified oligonucleotide;wherein the poly DNA spacer sequence in each of the first and secondchemically modified oligonucleotide is 3 or more nucleotide bases; andwherein each of the first and the second chemically modifiedoligonucleotide comprise a 3′-terminal chemical modification.
 13. Thekit of claim 12 wherein the kit further comprises a polymerase, anendonuclease for a nicking reaction, or both.
 14. The kit of claim 12wherein said poly DNA spacer of said first or second oligonucleotidecomprises from 3 to 20 bases.
 15. The kit of claim 13 wherein saidpolymerase has strand displacement activity, is 3′ to 5′ exonucleasedeficient, is 5′ to 3′ exonuclease deficient, or a combination thereof.16. The kit of claim 12 wherein said target nucleic acid is a micro-RNAor originates from an infectious agent.
 17. The kit according to claim12, further comprising instructions for use.
 18. The method of claim 1,further comprising detecting the presence or absence of at least onesignal DNA generated by at least one signal generation sequence.
 19. Themethod of claim 18, wherein detecting the presence of at least onesignal DNA indicates the presence of the target nucleic acid in thesample.
 20. The method of claim 1, wherein each of the first and thesecond oligonucleotide comprise a 3′-terminal chemical modification.